15N/14N and 18O/16O stable isotope ratios of nitrous oxide produced during denitrification in temperate forest soils

Anaerobic incubations of upland and wetland temperate forest soils from the same watershed were conducted under different moisture and temperature conditions. Rates of nitrous oxide (N2O) production by denitrification of nitrate (NO3) and the stable isotopic composition of the N2O (delta N-15, delta O-18) were measured. In all soils, N2O production increased with elevated temperature and soil moisture. At each temperature and moisture level, the rate of N2O production in the wetland soil was greater than in the upland soil. The N-15 isotope effect (epsilon) ((product - substrate)) ranged from -20 parts per thousand to -29 parts per thousand. These results are consistent with other published estimates of N-15 fractionation from both single species culture experiments and soil incubation studies from different ecosystems. A series of incubations were conducted with O-18-enriched water (H2O) to determine if significant oxygen exchange (O-exchange) occurred between H2O and N2O precursors during denitrification. The exchange of H2O-O with nitrite (NO2-) and/or nitric oxide (NO) oxygen has been documented in single organism culture studies but has not been demonstrated in soils prior to this study. The fraction of N2O-O derived from H2O-O was confined to a strikingly narrow range that differed between soil types. H2O-O incorporation into N2O produced from upland and wetland soils was 86% to 94% and 64% to 70%, respectively. Neither the temperature, soil moisture, nor the rate of N2O production influenced the magnitude of O-exchange. With the exception of one treatment, the net O-18 isotope effect (epsilon(net)) ((product substrate)) ranged from +37 parts per thousand to +43 parts per thousand. Most previous studies that have reported O-18 isotope effects for denitrification of NO3 to N2O have failed to account for the effect of oxygen exchange with H2O, When high amounts of O-exchange occur after fractionation during reductive O-loss, the O-18-enrichment is effectively lost or diminished and delta O-18-N2O values will be largely dictated by delta O-18-H2O values and subsequent fractionation. The process and extent of O-exchange, combined with the magnitude of oxygen isotope fractionation at each reduction step, appear to be the dominant controls on the observed oxygen isotope effect. In these experiments, significant oxygen isotope fractionation was observed to occur after the majority of water O-exchange. Due to the importance of O-exchange, the net oxygen isotope effect for N2O production in soils can only be determined using delta O-18 H2O addition experiments with delta O-18-H2O close to natural abundance. The results of this study support the continued use of delta N-15-N2O analysis to fingerprint N2O produced from the denitrification of NO3-. The utilization of O-18/O-16 ratios of N2O to study N2O production pathways in soil environments is complicated by oxygen exchange with water, which is not usually quantified in field studies, The oxygen isotope fractionation observed in this study was confined to a narrow range, and there was a clear difference in water O-exchange between soil types regardless of temperature, soil moisture, and N2O production rate. This suggests that O-18/O-16 ratios of N2O may be useful in characterizing the actively denitrifying microbial community. Crown copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.